This invention relates to modulating activities associated with the IL-9 pathway for the treatment of atopic allergies and related disorders such as asthma.
Inflammation is a complex process in which the body""s defense system combats foreign entities. While the battle against foreign entities may be necessary for the body""s survival, some defense systems respond to foreign entities, even innocuous ones, as dangerous and thereby damage surrounding tissue in the ensuing battle.
Atopic allergy is an ecogenetic disorder, where genetic background dictates the response to environmental stimuli. The disorder is generally characterized by an increased ability of lymphocytes to produce IgE antibodies in response to ubiquitous antigens. Activation of the immune system by these antigens leads to allergic inflammation and may occur after ingestion, penetration through the skin or after inhalation. When this immune activation occurs and is accompanied by pulmonary inflammation and bronchial hyperresponsiveness, this disorder is broadly characterized as asthma. Certain cells are important in this inflammatory reaction and they include T cells and antigen-presenting cells, B cells that produce IgE, basophils that bind IgE and eosinophils. These inflammatory cells accumulate at the site of allergic inflammation and the toxic products they release contribute to tissue destruction related to these disorders.
While asthma is generally defined as an inflammatory disorder of the airways, clinical symptoms arise from intermittent air flow obstruction. It is a chronic, disabling disorder that appears to be increasing in prevalence and severity (Gergen et al., 1992). It is estimated that 30-40% of the population suffer with atopic allergy and 15% of children and 5% of adults in the population suffer from asthma (Gergen et al., 1992). Thus, an enormous burden is placed on health-care resources.
Interestingly, while most individuals experience similar environmental exposures, only certain individuals develop atopic allergy and asthma. This hypersensitivity to environmental allergens known as xe2x80x9catopyxe2x80x9d is often indicated by elevated serum IgE levels or abnormally intense skin test response to allergens in atopic individuals as compared to nonatopics (Marsh et al., 1982). Strong evidence for a close relationship between atopic allergy and asthma is derived from the fact that most asthmatics have clinical and serologic evidence of atopy (Clifford et al., 1987; Gergen, 1991; Burrows et al., 1992; Johannson et al., 1972; Sears et al., 1991; Halonen et al., 1992). In particular, younger asthmatics have a high incidence of atopy (Marsh et al., 1982). In addition, immunologic factors associated with an increase in total serum IgE levels are very closely related to impaired pulmonary function (Burrows et al., 1989).
Both the diagnosis and treatment of these disorders are problematic (Gergen et al., 1992). The assessment of inflamed lung tissue is often difficult and frequently the source of the inflammation cannot be determined. It is now generally accepted that failure to control pulmonary inflammation leads to significant loss of lung function over time.
Current treatments suffer their own set of disadvantages. The main therapeutic agents, xcex2 agonists, reduce the symptoms thereby transiently improving pulmonary function, but do not affect the underlying inflammation so that lung tissue remains in jeopardy. In addition, constant use of xcex2 agonists results in desensitization which reduces their efficacy and safety (Molinoff et al., 1995). The agents that can diminish the underlying inflammation, such as anti-inflammatory steroids, have their own list of disadvantages that range from immunosuppression to bone loss (Molinoff et al., 1995).
Because of the problems associated with conventional therapies, alternative treatment strategies have been evaluated. Glycophorin A (Chu et al., 1992), cyclosporin (Alexander et al., 1992; Morely, 1992) and a nonapeptide fragment of interleukin 2 (IL-2) (Zavyalov et al., 1992) all inhibit potentially critical immune functions associated with homeostasis. What is needed in the art is a treatment for asthma that addresses the underlying pathogenesis. Moreover, these therapies must address the episodic nature of the disorder and the close association with allergy and intervene at a point downstream from critical immune functions.
In the related patent applications mentioned above, applicants have demonstrated that interleukin 9 (IL-9), its receptor and activities effected by IL-9 are the appropriate targets for therapeutic intervention in atopic allergy, asthma and related disorders.
Mediator release from mast cells by allergen has long been considered a critical initiating event in allergy. IL-9 was originally identified as a mast cell growth factor and it has been demonstrated that IL-9 up-regulates the expression of mast cell proteases including MCP-1, MCP-2, MCP-4 (Eklund et al., 1993) and granzyme B (Louahed et al., 1995). Thus, IL-9 appears to serve a role in the proliferation and differentiation of mast cells. Moreover, IL-9 up-regulates the expression of the alpha chain of the high affinity IgE receptor (Dugas et al., 1993). Elevated IgE levels are considered to be a hallmark of atopic allergy and a risk factor for asthma. Furthermore, both in vitro and in vivo studies have shown IL-9 to potentiate the release of IgE from primed B cells (Petit-Frere et al., 1993).
There is substantial support for the role of IL-9 gene in asthma. First, linkage homology between humans and mice suggests that the same gene is responsible for producing biologic variability in response to antigen in both species. Second, differences in expression of the murine IL-9 candidate gene are associated with biologic variability in bronchial responsiveness. In particular, reduced expression of IL-9 is associated with a lower baseline bronchial response in B6 mice. Third, recent evidence for linkage disequilibrium in data from humans suggests IL-9 may be associated with atopy and bronchial hyperresponsiveness consistent with a role for this gene in both species (Doull et al., 1992). Moreover, a genetic alteration in the human gene appears to be associated with loss of cytokine function and lower IgE levels. Fourth, the pleiotropic functions of this cytokine and its receptor in the allergic immune response strongly support a role for the IL-9 pathway in the complex pathogenesis of asthma. Fifth, in humans, biologic variability in the IL-9 receptor also appears to be associated with atopic allergy and asthma. Finally, despite the inherited loss of IL-9 receptor function, these individuals appear to be otherwise healthy. Thus, nature has demonstrated in atopic individuals that the therapeutic down-regulation of IL-9 and IL-9 receptor genes or genes activated by IL-9 and its receptor is likely to be safe.
While the role of the IL-9 gene, its receptor and their functions in atopic allergy, asthma and related disorders has been elucidated, a specific need in the art exists for elucidation of the role of genes which are regulated by IL-9 in the etiology of these disorders. Furthermore, most significantly, based on this knowledge, there is a need for the identification of agents that are capable of regulating the activity of these genes or their gene products for treating these disorders.
Cystic fibrosis is yet another disease which effects the lung and is associated with thick secretions resulting in airway obstruction and subsequent colonization and infection by inhaled pathogenic microorganisms (Eng et al., 1996). Cystic fibrosis airway epithelia exhibit a spectrum of ion transport properties that differ from normal, including not only defective cAMP-mediated chloride secretion, but also increased sodium absorption and increased calcium-mediated chloride secretion (Johnson et al., 1995). The increase in calcium-mediated chloride secretion is presumably an attempt to compensate for the overall decrease in chloride secretion due to the defect in cAMP-mediated chloride secretion. It does not adequately compensate for this defect, however, because normal chloride gradients are not maintained. Thus, potential therapeutic remedies for cystic fibrosis rely on mechanisms which increase chloride secretion in airway epithelial cells to compensate for defective cAMP-mediated chloride secretion. Such mechanisms are capable of restoring the cellular chloride gradient thereby alleviating sodium hyperabsorption associated with decreased chloride secretion. A specific need in the art therefore exists for identification of agents capable of enhancing calcium-dependent chloride secretion in cystic fibrosis airway epithelial cells.
The present invention includes new genes from the calcium activated chloride channel gene family designated ICACC (IL-9 Induced Calcium Activated Chloride Channel), particularly the mouse (SEQ ID NO:1) and human (SEQ ID NO:3 and SEQ ID NO:5) ICACC genes. The ICACC-1 genes are selectively up-regulated by IL-9 and therefore part of the IL-9 signaling pathway. The present invention also includes the protein products of the ICACC genes, particularly, the mouse (SEQ ID NO:2) and human (SEQ ID NO:4 and NO:6) ICACC genes.
The inventors have satisfied the need for diagnosis and treatment of atopic allergy, asthma and related disorders by demonstrating the role of ICACC-1 in the pathogenesis of these disorders. Therapies for these disorders are derived from the down-regulation of ICACC-1 as a member of the IL-9 pathway.
The identification of ICACC-1 has led to the discovery of compounds that are capable of down-regulating its activity. Activity is defined here as any alteration in either chloride channel function or expression of ICACC-1. Molecules that down-regulate ICACC-1 are therefore part of the invention. Down-regulation is defined here as a decrease in activation, function or synthesis of ICACC-1, its ligands or activators. It is further defined to include an increase in the degradation of ICACC-1 gene, its protein product, ligands or activators. Down-regulation is therefore achieved in a number of ways. For example, administration of molecules that can destabilize the binding of ICACC-1 with its ligands. Such molecules encompass polypeptide products, including those encoded by the DNA sequences of the ICACC-1 gene or DNA sequences containing various mutations. These mutations may be point mutations, insertions, deletions or spliced variants of the ICACC-1 gene. This invention also includes truncated polypeptides encoded by the DNA molecules described above. These polypeptides being capable of interfering with interaction of ICACC-1 with its ligand and other proteins.
A further embodiment of this invention includes the down-regulation of ICACC-1 function by altering expression of the ICACC-1 gene, the use of antisense gene therapy being an example. Down-regulation of ICACC-1 expression is accomplished by administering an effective amount of antisense oligonucleotides. These antisense molecules can be fashioned from the DNA sequence of the ICACC-1 gene or sequences containing various mutations, deletions, insertions or spliced variants. Another embodiment of this invention relates to the use of isolated RNA or DNA sequences derived from the ICACC-1 gene. These sequences containing various mutations such as point mutations, insertions, deletions or spliced variant mutations of ICACC-1 gene and can be useful in gene therapy.
Molecules that increase the degradation of the ICACC-1 protein may also be used to down-regulate its functions and are within the scope of the invention. Phosphorylation of ICACC-1 may alter protein stability, therefore kinase inhibitors may be used to down-regulate its function. Down-regulation of ICACC-1 may also be accomplished by the use of polyclonal or monoclonal antibodies or fragments thereof directed against the ICACC-1 protein. Such molecules are within the claimed invention. This invention further includes small molecules with the three-dimensional structure necessary to bind with sufficient affinity to block ICACC-1 interactions with its ligands or block function of the chloride channel. ICACC-1 blockade resulting in deregulation of calcium and chloride flux and other processes of proinflammatory cells where it is expressed make these small molecules useful as therapeutic agents in treating inflammation associated with atopic allergy, asthma and related disorders. In a further embodiment, aminosterol compounds are assessed for their ability to block ICACC-1 induction by IL-9 or antigen as a means of determining their usefulness in treating atopic allergies and related disorders.
The agents discussed above represent various effective therapeutic compounds in treating atopic allergies, asthma and other related disorders. Applicants have thus provided antagonists and methods of identifying antagonists that are capable of down-regulating ICACC-1. Applicants also provide methods for down-regulating the activity of ICACC-1 by administering truncated protein products, chloride channel blockers, aminosterols and the like.
Applicants also provide a method for the diagnosis of susceptibility to atopic allergy, asthma and related disorders by describing a method for assaying the induction of ICACC-1, its functions or downstream activities. In a further embodiment, Applicants provide methods to monitor the effects of ICACC-1 down-regulation as a means to follow the treatment of atopic allergy and asthma. Applicants also provide a method for diagnosing autoimmune type diseases such as inflammatory bowel disease (IBD) where suppression of TH2-associated responses (such as the biologic responses associated with IL-9) are a common molecular feature. The constitutive expression of ICACC-1 in the small intestine and colon suggest that this is a useful marker for monitoring treatment of TH1 associated disease states such as IBD, where down regulated expression of ICACC-1 will be associated with the disease.
In a further embodiment, Applicants identify a disease state, which can be treated through the up-regulation of ICACC-1. Applicants provide a method for treating the defect in cAMP-mediated chloride secretion in cystic fibrosis airway epithelia by further increasing calcium-dependent chloride secretion through up-regulation of ICACC-1. This up-regulation of ICACC-1 resulting in increased chloride secretion and thus restoration of the cellular chloride gradient resulting in normal airway epithelial cell function. Applicants provide a method for treating inflammatory bowel disease (IBD) with local delivery of ICACC-1 via gene therapy or up regulation of ICACC-1 to enhance TH2-associated responses for suppressing the T1-associated IBD autoimmune disease.
The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description, serve to explain the principle of the invention.